There is a wealth of information about peripheral steroid synthesis and secretion, but surprisingly little information about actual concentrations of steroids in active neural circuits. This gap in our knowledge of brain steroids, especially those synthesized in the brain (neurosteroids), hampers efforts to identify optimal treatments to improve human mental health and cognition. Estradiol (E2) can enhance memory function in humans and non-human animal models by interactions with hippocampal glutamatergic and GABAergic signaling, but the mechanisms are poorly understood. Because the hippocampus synthesizes E2 de novo, perhaps at synaptic terminals, measures of peripheral E2 concentrations tell us little about biologically active local levels. Real-time in vivo measures of hippocampal E2 levels would be of great value, especially given evidence for rapid regulation of steroid synthetic enzymes and for rapid actions of E2 on memory function. To address this gap we have developed the ability to measure steroid levels in freely-behaving songbirds using in vivo microdialysis. Our findings show that E2 and testosterone (T) levels in the songbird brain fluctuate relatively rapidly during social interactions independent of their peripheral levels or levels in adjacent brain regions; inhibition of this E2 flux has clear behavioral implications. Further, we find that E2 naturally fluctuates inversely relative to glutamate and retrodialysis of glutamate rapidly reduces E2 concentrations. How and why these fluctuations occur is unknown. We propose performing microdialysis on the songbird hippocampus to measure steroids, glutamate and GABA as the birds perform natural hippocampal-based spatial and temporal memory intensive behaviors. We will use retrodialysis to inhibit neurosteroid synthesis and examine spatial-memory performance. We will combine microdialysis with brain extraction procedures to determine the biologically active neuroestrogen concentrations. We will identify mechanisms whereby neurotransmitters regulate steroidogenic enzymes in hippocampal slices of brain to produce the levels we see in vivo. Results of these studies will help clarify important concepts about steroids as neuromodulators/neurotransmitters and will help resolve debate over the role of estrogens on specific aspects of cognition, hippocampal physiology and memory function. Therapies that impact hippocampal estrogen signaling may provide relief from disorders such as post-traumatic stress and cognitive impairment arising from psychiatric disorders such as depression and psychosis.